Purification and Properties of Two 2‐Oxoacid:Ferredoxin Oxidoreductases from Halobacterium halobium

Abstract

Pyruvate: ferredoxin oxidoreductase and 2‐oxoglutarate: ferredoxin oxidoreductase were obtained from cell‐free extracts of Halobacterium halobium as homogeneous proteins after ammonium sulfate precipitation, salting‐out chromatography with ammonium sulfate on unsubstituted agarose, gel filtration and chromatography on hydroxyapatite. The respective molecular weights are 256000 and 248000. Both enzymes consist of two sets of nonidentical subunits of Mr 86000 and 42000 in the case of the pyruvate‐degrading enzyme and of 88000 and 36000 in the case of the 2‐oxoglutarate‐degrading enzyme. Analyses indicate that an intact enzyme molecule contains two [4 Fe‐4S]2+(2+.1+) clusters and two molecules of thiamin diphosphate. Flavin nucleotides, lipoic acid and pantetheine are absent. Thus the enzymes are very similar to the 2‐oxoacid: ferredoxin oxidoreductases from fermentative and photosynthetic anaerobes described previously, but are clearly different from the 2‐oxoacid dehydrogenase multienzyme complexes which commonly occur in aerobic organisms.

Thermoacidophilic Archaebacteria Contain Bacterial-Type Ferredoxins Acting as Electron Acceptors of 2-Oxoacid: Ferredoxin Oxidoreductases

“…Acyl thioesters of coenzyme A are formed as well, but ferredoxin acts as the electron acceptor. More recently these enzymes have also been found in some species of cyanobacteria [5,6] and in extreme halophiles [7]. The only common property of 2-0x0-acid dehydrogenase complexes and 2-oxoacid : ferredoxin oxidoreductases in the presence of the prosthetic group thiamin diphosphate.…”

“…Desulfurococcus ferredoxin was eluted from DEAE-cellulose at the same ionic strength as Thermoplasma ferredoxin. The redox centres in archaebacterial2-oxoacid : ferredoxin oxidoreductases, which in Halobacterium were identified as [4Fe-4S]2+'2+s'+' clusters [7], are able to transfer electrons to horse heart cytochrome c without the action of ferredoxin. While this represents only a minor contribution to the total electron transfer activity of Halobacterium enzymes, ferredoxin-free extracts of Thermoplasma and Sulfolobus still reduce cytochrome c at a considerable rate.…”

“…It remains stable until transfer of the acyl moiety to CoASH occurs [8]. Thus 2-oxoacid oxidation is achieved by two steps of single-electron transfer [9].…”

Molecular mechanism of pyruvate‐ferredoxin oxidoreductases based on data obtained with the Clostridium pasteurianum enzyme

“…The quaternary structure and partial sequence of C. pasteurianum PFO resemble those of similar enzymes from bacteria or unicellular eucarya [4,[13][14][15][16], but differ from the heterodimers or heterotetramers found in archaea [3,10,17,18].…”

“…Several other c~-ketoacids with 5 (~-ketovalerate, aketoisovalerate, c~-ketoglutarate) or 6 carbon atoms (~-ketocaproate, c~-ketoisocaproate) were not substrates of the enzyme. Indeed, oxidative decarboxylation of other 2-oxoacids exists but it is carried out by other enzymes [10]. Clostridial ferredoxins are the type of electron acceptor displaying the highest specificity for the enzyme [11].…”